This project will characterize biochemical aspects of acetylcholine (ACh) storage by mammalian brain synaptic vesicles. Bovine brain will be used as starting material for isosmotic isolation of highly purified synaptic vesicles of mixed neurotransmitter type using biochemical separation techniques routinely used for purification of Torpedo electric organ synaptic vesicles. In Torpedo vesicles it is known that the ACh storage system is composed of an ATPase, s transporter for ACh and a receptor for the inhibitory compound 2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183). Binding of (3H)vesamicol to the brain receptor in purified vesicles will be characterized, including determining whether receptor can exist in a cryptic form as in Torpedo vesicles. The measurements will be obtained using a vacuum assisted filtration technique. Radiolabeled ACh active transport by the purified brain vesicles will be studied also by the filter method. The linkage between (3H)vesamicol binding and (14C)ACh active transport inhibition in the same samples will be characterized to see if it is as complex as in Torpedo vesicles. The vesamicol receptor of brain vesicles will be detergent solubilized and purified to homogeneity to determine how similar its composition is to that of the Torpedo receptor. Finally, the ACh transporter in brain vesicles will be photoaffinity-labeled with a radioactive ACh analogue and the labeled subunit identified by gel electrophoresis and fluorography. The overall aim of this project is to determine the extent of similarity of the ACh storage system in brain to that of the electric organ. The long term aim is to understand the biochemistry of ACh transport and storage, and to determine the relationship of ACh transport to ACh release in the mammalian cholinergic nerve terminal. This includes an understanding of all the regulatory features in the system so that they might be pharmacologically or otherwise manipulated to increase ACh storage in and release from compromised cholinergic terminals. Since many disease or toxic states impact selectively on the cholinergic nervous system of humans, for example, Alzheimer's disease, this capability could be of significant clinical benefit.